Epidemiology of Nontuberculous Mycobacteria in Tuberculosis suspects, Southwest of China, 2017-2022

Objectives This study summarizes the epidemiological characteristics, species distribution, and drug sensitivity of clinical nontuberculous mycobacteria (NTM) isolates at the Public Health Clinical Center of Chengdu, China, from January 2017 to December 2022. Methods We retrospectively analyzed data from patients with clinically isolated NTM strains. Chi-square analysis assessed the rate of Mycobacterium strain isolation over 6 years. Results The number of samples tested for Mycobacterium tuberculosis (MTB) and/or NTM increased each year, while MTB detection decreased and NTM detection rose significantly each year (P=0.03). The average age of NTM patients was 51 ± 17.53 years, with a 14.1% HIV infection rate. The predominant isolates were Mycobacterium avium-intracellulare (MAC) and M. chelonae/M. abscessus, with 96.4% of cases being of Han ethnicity. Amikacin, moxifloxacin, and clarithromycin were effective against M. avium and M. intracellulare; linezolid, amikacin, and cefoxitin were effective against M. chelonae/M. abscessus. Over 90% of NTM cases originated from the respiratory tract. Conclusion The NTM isolation rate in Southwest China has risen in recent years, primarily among elderly patients with a high HIV co-infection rate. The main NTM isolates were MAC and M. chelonae/M. abscessus. Amikacin, moxifloxacin, clarithromycin, and linezolid exhibited strong antibacterial activity against SGM, while amikacin and linezolid displayed relatively better antibacterial activity against RGM. The prevalence of NTM infection may be positively associated with regional economic development and health conditions.


Introduction
Nontuberculous mycobacteria (NTM) are part of the Mycobacterium species, distinct from those in the M. tuberculosis complex or M. leprae, and typically act as opportunistic pathogens (Falkinham, 1996;Falkinham, 2002).This term encompasses around 200 distinct Mycobacterium species, with new species continually emerging (Zhou et al., 2020).In recent years, both in China and globally, NTM infections have been on the rise (Wang et al., 2019;Liu et al., 2021;Thornton et al., 2021).Among clinical laboratories in China, the Mycobacterium avium-intracellulare complex (MAC) and the M. chelonae/M.abscessus complex are the two most commonly encountered NTM complexes, and they are among the most drugresistant species (Wang et al., 2016;Wang et al., 2019;Guo et al., 2021).However, limited studies have explored NTM drug susceptibility due to small sample sizes or restricted antibiotic types being tested.Therefore, this study aims to summarize NTM identification and drug sensitivity data from the largest sample size in a major central city in Southwest China.This information can provide valuable insights for the prevention and treatment of NTM diseases.

Laboratory quality control
External quality assessment (EQA) was carried out for smear, culture, and DST at the Innovation Alliance for TB Diagnosis and Treatment in Beijing, China.Additionally, a blinded retesting of approximately 10% of isolates from the study laboratory was conducted by a specialized Centers for Disease Control and Prevention.

Statistical analysis
Data were analyzed using SPSS Statistics Client 19.0 (SPSS Inc., IL, USA).Normally distributed measurement data were presented as means, while categorical variables were expressed as numbers and percentages.Chi-square (c 2 ) analysis was employed to assess variations in the Mycobacterium strain rate, age, and sex ratio over five years.Statistical significance was set at P < 0.05.

Ethics approval and consent to participate
This study received approval from the Ethics Committee of PHCC (Approval No. 2017Y025).All patient information used in this study was routinely collected through the mandatory notification system.The requirement for informed consent was waived by the ethics committee.

Demographic and clinical characteristics
From January 2017 to December 2022, a total of 126,368 nonrepeated clinical specimens with suspected mycobacterial infections were cultured at PHCC.Among these specimens, 26,510 (21.0%) tested positive in culture.MTB was detected in 25,923 (97.8%) positive samples, while NTM was found in 587 (2.2%) samples.Among all NTM cases, 13 were co-infected with both MTB and NTM (Figure 1; Table 1).Over the 6-year study period, a significant increase was observed in the number of samples tested for MTB and/or NTM each year, corresponding to a significant annual rise in NTM detection (c 2 = 18.01,P=0.03; Figure 1).The mean age of the 587 NTM patients was 51 ± 17.53 years (range: 13-88 years).Within this subgroup, 349/587 (59.5%) were males, and 238/587 (40.5%) were females, with a male-to-female ratio of 1.5.There was no significant difference in the male-to-female ratio over the 6-year period (c 2 = 0.71, P>0.05; Figure 2).The majority of NTM-infected cases were middle-aged and elderly patients, and the proportion of each age group showed no significant difference over the 6 years (c2 = 11.16,P>0.05; as shown in Figure 2; Table 1).
Regarding the Rapidly Growing Mycobacteria (RGM), linezolid, amikacin, and cefoxitin were the most effective agents against M. chelonae/M.abscessus.Rifampicin, clarithromycin, linezolid, amikacin, and moxifloxacin showed complete sensitivity in two strains of M. lentiflavum, while linezolid, amikacin, cefoxitin, and moxifloxacin demonstrated complete sensitivity in three strains of M. fortuitum.

Discussion
According to recent reports from China and other countries, there has been a gradual increase in the incidence of NTM infection and laboratory isolation rates (Wang et al., 2016;Wang et al., 2019;Guo et al., 2021;Liu et al., 2021;Thornton et al., 2021).NTMs are inherently resistant to many anti-TB drugs (ATDs), and treatment plans depend on factors such as the NTM species, infection site, and the severity of the infection (Lee et al., 2015;Daley et al., 2020;Gopalaswamy et al., 2020).The prevalence of NTM strains varies among different regions and populations.For instance, the most common NTM isolates reported in various countries include M. kansasii and MAC in Poland (Przybylski et al., 2023) In southwest China, there has been limited data on NTM infections.Our preliminary study discussed only a small sample of NTM-infected individuals in Southwest China, while a few studies have reported on large samples of clinically NTM-infected individuals and the dynamic sensitivity of NTM to multiple antibiotics in China and worldwide (Jeong et al., 2017;Zhou et al., 2020;Maya et al., 2022).In this study, we conducted a systematic analysis of NTM clinical infection cases in Southwest China over the past six years.The results indicated a rising trend in the number of patients visiting PHCC for mycobacteria culture evaluation each year from 2017 to 2022.This trend may be attributed to increased public awareness of healthcare in recent years and the expanding reach of PHCC in Southwest China.However, in 2020, there was a slight decline in this trend, likely due to the initial COVID-19 pandemic control measures, which resulted in reduced public mobility and hospital visits.Nevertheless, the trend resumed its upward trajectory after 2021.Moreover, among mycobacteria-positive cultures, the count of MTB cases increased from 2017 to 2019 and then gradually decreased until 2020.This trend is consistent with the recent control measures for TB, which have led to decreasing incidence and mortality rates each year (World Health Organization, 2021).Notably, the number of isolated clinical NTM cases showed a continuous upward trend from 2017 to 2022, rising from 11 cases in 2017 to 203 cases in 2022.This finding aligns with reports of increasing NTM infection cases worldwide and the observation that laboratory isolation rates have been on the rise each year (Loṕez-Roa et al., 2020).
In our previous study, we observed that MTB infections accounted for a significant proportion of ethnic minorities, particularly among children with TBM in Southwest China (Wang et al., 2020).However, in this study, 96.4% of NTMinfected patients were of Han ethnicity from central cities, with minority groups representing a small subset of the infected individuals.This pattern contrasts with the MTB-infected population model in southwest China, suggesting a positive correlation between NTM infection and the regional level of economic development and sanitation conditions.
Among the 587 NTM cases in this study, the top three isolated strains were M. avium, M. chelonae/M.abscessus, and M. intracellulare, followed by M. kansasii.These clinical strains mirrored the domestic epidemic trend (Jing et al., 2012;Wu et al., 2014;Tan et al., 2018;Liu et al., 2019).Among the 113 cases that underwent in vitro drug sensitivity testing, 90.3% of the strains originated from the respiratory tract, followed by cerebrospinal fluid, lymph nodes, the gastrointestinal tract, and pleural effusion.Intriguingly, MAC and M. chelonae/M.abscessus were the predominant strains in various tissues, followed by M. kansasii, M. fortuitum, M. scrofulaceum, M. gordoniae, and M. lentiflavum.
NTM naturally exhibits resistance to various anti-TB drugs, and its clinical isolation rate is relatively low.Furthermore, the emergence of NTM resistance poses a significant challenge in clinical treatment.Currently, research on NTM drug resistance is limited, with only a few reports on the sensitivity of NTM to various antibiotics in China, often based on small sample sizes or focused on a single antibiotic (Lan et al., 2011;Li et al., 2017).In this study, we conducted comprehensive in vitro sensitivity tests on 113 NTM strains with 13 antibiotics.The results revealed that clarithromycin exhibited significant antibacterial efficacy against M. avium and M. intracellulare, with drug resistance rates of 17.2% and 22.2%, respectively.The average drug resistance rate among SGM was 17.8%, while for RGM, it was 62.5%.Aminoglycosides are commonly used and effective drugs for NTM treatment.In our study, amikacin demonstrated potent antibacterial effects on M. avium and M. intracellulare, with low drug resistance rates of 6.9% and 18.5%, respectively.The antibacterial activity of amikacin against MAC was superior to that of clarithromycin.Notably, amikacin also displayed good antibacterial efficacy against both SGM and RGM, with drug resistance rates of 13.7% and 45.0%, respectively.In addition to its effectiveness against MAC, tobramycin's antibacterial activity against M. chelonae/M.abscessus was notable at 94.3%, although it differed from amikacin at 51.4%.This discrepancy may be attributed to variations in ethnicity, geographical regions, or clinical isolates.While aminoglycosides demonstrate potent antibacterial activity against NTM, their prolonged use can lead to hepatorenal toxicity and ototoxicity.Therefore, these drugs should be used judiciously in clinical practice.Additionally, in this study, the fluoroquinolone moxifloxacin exhibited robust antibacterial activity against SGM but showed weaker effectiveness against RGM.

Conclusions
The isolation rate of NTM in southwest China has shown an increasing trend in recent years.The majority of infected cases involve elderly patients, and there has been an elevated proportion of individuals with HIV infection.The predominant clinical isolates are MAC and M. chelonae/M.abscessus, followed by M. kansasii and M. fortuitum.Among the tested antibiotics, amikacin, moxifloxacin, clarithromycin, and linezolid demonstrated effective antibacterial activity against SGM, whereas linezolid and amikacin exhibited relatively better antibacterial activity against RGM.The incidence of NTM infection may be positively correlated with the level of regional economic development and healthcare conditions.

Limitations
One significant limitation of this study is the extended treatment cycle required for NTM infections (Lee et al., 2015;Daley et al., 2020;Gopalaswamy et al., 2020), often exceeding one year or even longer.Consequently, many cases experience issues such as loss to follow-up, poor treatment outcomes, and a high recurrence rate.Although follow-up observations are ongoing for some cases to assess treatment efficacy, the data collection for these cases is incomplete.Future research should aim to provide more comprehensive and valuable reference information for clinical use.
This study included all patients with culture-positive NTM infections treated at the Public Health Clinical Center of Chengdu (PHCC) in Sichuan Province, China, from January 2017 to December 2022.During the 6-year study period, a total of 126,368 suspected mycobacterial infections were cultured using the BACTEC ™ MGIT 960 System (Becton Dickinson & Co., NJ, USA), resulting in 26,510 Mycobacterium tuberculosis (MTB) culture-positive cases and 587 NTM culture-positive cases.Diagnosis and categorization of NTM-infected patients were based on the 2012 and 2020 NTM Diagnosis and Treatment Expert Consensus (Editorial Board of CSTB, 2016; Tuberculosis Branch of Chinese Medical Association, 2020), the Clinical Diagnosis and Treatment Guidelines for Tuberculosis in China (Chinese Medical Association, 2005), and the updated guidelines from the World Health Organization.Diagnosis of human immunodeficiency virus (HIV) followed the Chinese HIV and HIV Infection Diagnostic Criteria (WS293-2008) (From the Centers for Disease Control and prevention, 1993).

FIGURE 2
FIGURE 2 Distribution of Male-to-Female Ratio and Proportion of Age in NTM Patients During 2017-2021.NTM, nontuberculous mycobacteria.
, MAC and M. gordonae in Italy(Giannoni et al., 2023), M. fortuitum, and M. simiae in Iran(Tarashi et al., 2023), and MAC, M. abscessus, and M. kansasii in Turkey(Babalik et al., 2023).In Switzerland, common NTM isolates include M. avium and M. gordonae (Vongthilath-Moeung et al., 2022).However, information regarding NTM isolates and drug resistance profiles in southwest China has been scarce.Therefore, accurately understanding the NTM epidemic and drug resistance situation is crucial for early differential diagnosis and treatment of TB and NTM diseases.

TABLE 1
General characteristics among patients with NTM disease (n=587).

TABLE 3
Number of Nontuberculous Mycobacteria clinical strains resistant to drugs in vitro experiments.